In the past several years immunoconjugates have been developed as an alternative therapeutic approach to treat malignancies. Immunoconjugates were originally composed of an antibody chemically conjugated to a plant or a bacterial toxin, a form that is known as an immunotoxin. The antibody binds to the antigen expressed on the target cell and the toxin is internalized causing cell death by arresting protein synthesis and inducing apoptosis (Brinkmann, U., Mol. Med. Today, 2:439-446 (1996)). More recently, genes encoding the antibody and the toxin have been fused and the immunotoxin expressed as a fusion protein.
A variety of plant, fungal, and bacterial toxins have been adapted for use with immunotoxins, including ricin, diphtheria toxin, and Pseudomonas exotoxin A (PE) (Pastan, I. et al., Nat Rev Cancer, 6:559-565 (2006); Pastan, I. et al., Annu Rev Med, 58:221-237 (2007)). PE-based immunotoxins are currently in clinical trials for the treatment of CD22-expressing lymphomas and leukemias, as well as mesothelin-expressing solid tumors (Kreitman, R., et al., J Clin Oncol, 23:6719-6729 (2005); Hassan, R., Clin Cancer Res, 13:5144-5149 (2007)). Typically, the PE has been truncated or mutated to reduce its non-specific toxicity while retaining its toxicity to cells to which it is targeted by the targeting portion of the immunotoxin. Over the years, numerous mutated and truncated forms of PE have been developed. The one used in most clinical trials to date is a 38 kD truncated form referred to as “PE38.”
Despite these decades of efforts, current PE-based immunotoxins are still not fully satisfactory. Although the PE38 immunotoxins that have reached clinical trials are comparatively well tolerated at low doses, dose-limiting toxicities have restricted their therapeutic effect. In a phase I clinical trial of a PE-based immunotoxin known as LMB-2, dose-limiting toxicities above 40 μg/kg given every other day (QOD) X 3 consisted of transaminase elevation, diarrhea, cardiomyopathy and an allergic reaction (Kreitman, R. J. et al., J Clin Oncol, 18:1622-1636 (2000)). In a phase I clinical trial of an anti-mesothelin immunotoxin, referred to as SS1P, adverse events of pleuritis, urticaria, and vascular leak syndrome were found to be dose limiting (Hassan, R. et al., Clin Cancer Res, 13:5144-5149 (2007)). In a phase I trial of a third PE-based immunotoxin, BL22, dose-limiting toxicities included several cases of hemolytic uremic syndrome and a cytokine release syndrome with systemic vascular leak syndrome (Kreitman, R. J. et al., J Clin Oncol, 23:6719-6729 (2005)).
Further, the PE-based immunotoxins currently in clinical trials are highly immunogenic. This has proven not to be a problem in the treatment of hematological malignancies, in which the ability of the immune system to mount a response is often compromised. Immunotoxins can typically be administered multiple times to patients with hematological malignancies. Patients with solid tumors, however, usually develop neutralizing antibodies to PE-based immunotoxins within weeks after the first administration. Since many protocols call for a three week period between administration of immunotoxins, the development of the antibodies during this period effectively means that, for solid tumors, usually only one administration can be made of a PE-based immunotoxin before the patient's antibodies render it ineffective. Even a single administration of a PE-based immunotoxin can be highly useful in reducing the patient's tumor burden, in eliminating smaller metastases, and in alleviating symptoms, but the ability to administer multiple doses would clearly be useful.
A limited number of approaches have been developed as an attempt to address these problems. One approach to reducing non-specific toxicity, reducing the isoelectric point of the framework regions of Fvs used as the targeting moiety of immunotoxins, was reported in co-owned PCT Application No. PCT/US01/43602, published as International Publication No. WO 02/40545. An approach to reducing immunogenicity is described in co-owned PCT application No. PCT/US06/028986, published as WO 2007/016150, which reports mapping of the various epitopes of PE and mutations of individual amino acid residues that could be combined to reduce the overall immunogenicity of the resulting PE molecule compared to that of PE38. Nonetheless, it would be desirable to have additional approaches of reducing the dose-limiting toxicity of the immunotoxin. Further, it would be desirable to have additional approaches for reducing the immunogenicity of PE and of immunotoxins in which PE acts as the toxic moiety. The present invention satisfies these and other needs.